PROJECT ABSTRACT The long-term goal of this project is to optimize a protein therapeutic for Duchenne muscular dystrophy (DMD), and potentially other muscle diseases, that will enhance the repair capacity of muscle cell membranes that are compromised by mutations in the dystrophin/dystroglycan complex. Mutations in the dystrophin/dystroglycan complex result in Duchenne muscular dystrophy. Myos Inc. is developing a novel recombinant construct of the tripartite protein 72/mitsugumin 53 (TRIM72/MG53), an essential regulator of membrane repair in skeletal and cardiac muscle. rhMG53 therapy ameliorates disease pathology in the dystrophin-null mouse model, strongly suggesting that it may enhance repair and restoration of muscle function in DMD. However, its large size and short serum half-life make rhMG53 unsuitable for protein therapy. Therefore, the objective of this Phase I STTR project is to engineer the rhMG53 protein, the result of which will be called MyoTRIM, for use in treating DMD by optimizing its functional and pharmacokinetic (PK) properties. This STTR project is a collaboration with Noah Weisleder, Ph.D. (Ohio State University), who is the PI. Aim 1 is to engineer a compact rhMG53 protein containing key moieties that are required for membrane repair. Deletion analysis and protein engineering approaches will be used to selectively delete regions that are not predicted to affect protein folding. This will reduce protein size from the current 53 kDa to <24 kDa while retaining the essential functional domains and 3D structure. Aim 2 is to improve the PK characteristics of the engineered rhMG53 protein by PEGylation. A panel of three candidate rhMG53 construct resultant from Aim 1 will be modified by covalent and/or non-covalent attachment of polyethylene glycol (PEG) in order to extend its serum half-life from the current 4 hr to >12 hr. Protein function will be tested in an ex vivo membrane repair assay and in the D2.B10 (DBA/2-congenic) Dmdmdx (D2- mdx) mouse model of DMD. Outcome measures will include serum half-life, protein concentrations in serum and target tissues, and serum biomarkers for skeletal and cardiac muscle membrane integrity. This novel, truncated, functional PEGylated rhMG53 construct has great potential to improve muscle membrane repair to treat fatal muscular dystrophies and other forms of muscle disease, independent of gene or mutation class. By enabling membrane resealing and preservation of skeletal and cardiac muscle function, it would provide a complementary treatment approach to other therapeutic efforts now in development. It also may provide a platform technology to target other diseases involving compromised membrane integrity or necrotic cell death, such as cardiovascular disease and neurodegenerative disorders. Successful completion of this Phase I STTR project will result in a novel rhMG53-PEG that is suitable for therapeutic development.